Journal of Chromatographic Science, 2016, Vol. 54, No. 3, 305–311
doi: 10.1093/chromsci/bmv142
Advance Access Publication Date: 24 October 2015
Article
Article
Validation of HPLC–UV Assay of Caffeic Acid
in Emulsions
Caroline Magnani Spagnol, Vera Lucia Borges Isaac,
Marcos Antonio Corrêa*, and Hérida Regina Nunes Salgado
Department of Drugs and Medicines, Faculty of Pharmaceutical Sciences of Araraquara, Universidade Estadual
Paulista “Júlio de Mesquita Filho”—UNESP, Araraquara, São Paulo, Brazil
*Author to whom correspondence should be addressed. Email: [email protected]
Received 20 March 2015; Revised 9 July 2015
Abstract
An accurate, sensitive, precise and rapid reversed-phase high-performance liquid chromatographic
method was successfully developed and validated for the determination of caffeic acid (CA) in emulsions. The best separation was achieved on a 250 × 4.6 mm, 5.0 µm particle size RP18 XDB Waters
column using ethanol and purified water (40:60 v/v) adjusted to pH 2.5 with acetic acid as the mobile
phase at a flow rate of 0.7 mL/min. Ultraviolet detection was performed at 325 nm at ambient column
temperature (25°C). The method was linear over the concentration range of 10–60 µg/mL (r 2 = 0.9999)
with limits of detection and quantification of 1.44 and 4.38 µg/mL, respectively. CA was subjected to
oxidation, acid, base and neutral degradation, as well as photolysis and heat as stress conditions.
There were no interfering peaks at or near the retention time of CA. The method was applied to
the determination of CA in standard and pharmaceutical products with excellent recoveries. The
method is applicable in the quality control of CA.
Introduction
Caffeic acid (CA) (Figure 1) (C9H8O4, 3,4-dihydroxycinnamic acid,
molecular mass 180.16 g/mol, CAS number 331-39-5) occurs as a
light yellow powder. CA is partially soluble in cold water and is readily
soluble in hot water and ethanol. The functional groups corresponding to the pKa values of CA are shown in Figure 2 (1).
CA is one of the most widely distributed hydroxycinnamate and
phenylpropanoid metabolites in plant tissues. This polyphenol is present
in blueberries, coffee, cider and apples (2). Besides food, CA is also present in propolis and is used in several widely used medications (3). CA
acts as a carcinogenesis inhibitor (4,5) and is also known to exert antioxidant and antibacterial effects in vitro and can contribute to the prevention of atherosclerosis and other cardiovascular diseases (6–8).
Owing to its high antioxidant activity, CA protects organisms
from the deleterious effects of free radicals and can be used in cosmetic
emulsions for dermal application in order to maintain healthier and
younger looking skin by preventing skin dehydration, pigmentation,
fine wrinkles, sagging and neoplasm diseases (1).
Emulsions are widely used to administer drugs or active cosmetic
components topically owing to their numerous advantages. Emulsified
preparations tend to be more acceptable to users because of ease of application and good spread ability on the skin (9–11). However, emulsion systems are thermodynamically unstable since they are formed by
complex mixtures of a wide variety of excipients with nonpolar or
polar characteristics that are immiscible with each other and must be
stabilized by an emulsifying surfactant (12–14).
During the development of a cosmetic emulsion, in addition to the
aesthetic appearance, the physical and chemical properties of the formulation are critical (15) because stability of the emulsion may be affected by temperature, transport and storage time (16).
The development of effective analytical methods for quality control of marketed drugs is extremely important and aims to provide reliable information about the nature and composition of the materials
under analysis (17). There is no method for determination and quantification of CA in official compendia, such as pharmacopoeias; however, most current articles recommend quantification of CA by
high-performance liquid chromatography (HPLC). Table I lists some
methods for the determination of CA.
Quality control in the pharmaceutical and cosmetic industries requires reliable analytical methods for ensuring therapeutic efficacy of
© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
305
306
Spagnol et al.
Rheodyne Breeze 7725i manual injector and a Waters 2487
ultraviolet-visible (UV-Vis) detector.
Figure 1. Chemical structure of CA.
Figure 2. Functional groups corresponding to pKa values of CA: 12:79 pKa1 ;
9:97 pKa2 and 4:04 pKa3 .
the marketed drug/substance; these methods must also be simple, fast
and versatile and should not have complex or costly procedures or
generate toxic wastes to facilitate their use in laboratory tests. The
HPLC–UV method has been employed for compound identification
by comparison of the spectral profile with that of standard materials;
this feature is important for characterization of a substance by determination of the absorbance maxima (29). The use of this method for
quantification of drugs has earned distinction owing to its favorable
characteristics for application in quality control. The inclusion of
this instrumental technique in official textbooks is highly recommended because of the accuracy and reproducibility of HPLC–UV data (30).
As an alternative to the existing methods, herein, an inexpensive,
useful, fast and simple HPLC–ultraviolet (UV) method has been developed, validated and applied to the quantitative determination of CA in
emulsions with the objective to apply this technique in routine analysis
in the pharmaceutical industry to ensure therapeutic efficacy of drugs
already marketed globally. In addition to the aforementioned advantages, this method uses only green solvents and reduces toxic waste
generation.
Experimental
Instrumentation and reagents
Materials and standards
HPLC grade ethanol (Synth, Brazil) and HPLC grade acetic acid (J.T.
Baker®, USA) were used. Deionized MilliQ water (Millipore, Bedford,
MA) was used to prepare the mobile phase and diluent solutions. The
CA standard was supplied by Sigma-Aldrich, and the emulsion was
prepared with CA from Nanjing Zelang Medical Technology. The
mobile phase was prepared by mixing ethanol and purified water
(40:60 v/v; adjusted to pH 2.5) with acetic acid and filtering through
a 0.45-µm membrane filter. The diluent solution was prepared by mixing ethanol and purified water (40:60 v/v). A stock standard solution
equivalent to 200 µg/mL CA was prepared by dissolving an accurately
weighed amount of pure substance in the diluent solution.
Equipment
HPLC analysis was performed on a Waters HPLC system equipped
with a Waters 1525 binary gradient chromatography pump, a
Chromatographic conditions
Chromatographic analysis was conducted at ambient temperature
(25°C) on an RP18 XDB Waters column (250 × 4.6 mm, 5.0 µm particle size). The mobile phase comprised a mixture of ethanol and purified water (40:60 v/v) adjusted to pH 2.5 with acetic acid. The flow
rate was 0.7 mL/min. The detector wavelength was set to 325 nm, and
the injection volume was 10 µL.
Sample
An emulsion (100 g) was prepared by the method generally used in the
industrial process. CA (1 g) was incorporated into the oil phase of the
cream (Table II). An aliquot of this emulsion was diluted with 25 mL
of ethanol and filtered through a 0.45-µm membrane. This solution
was diluted in a ratio of 1:5 with the mobile phase to achieve the required final CA concentration of 40 µg/mL. After filtration through a
regenerated cellulose membrane (0.45 µm), the extracted CA solution
was analyzed by HPLC.
Methods
Method validation
Method validation was performed as stipulated in the International
Conference on Harmonization (ICH) specifications (31) for linearity,
selectivity, accuracy, precision, robustness, detection limit and quantitation limit.
Linearity
Ten microliters of working standard solutions (10–60 µg/mL CA)
were manually injected onto the column in triplicate. The areas used
in determining the calibration curve were statistically evaluated by
analysis of variance (ANOVA). The equation of the line was determined by linear least squares regression analysis.
Selectivity
The selectivity was evaluated by a forced degradation procedure. CA
solutions were prepared in 1 × 10−3 mol/L HCl, 1 × 10−3 mol/L
NaOH, 3% H2O2 and H2O at a concentration of 40 µg/mL, and all
four solutions were kept in a hot water bath at 80°C. Aliquots of these
solutions were withdrawn at intervals of 2, 4 and 8 h and analyzed immediately using HPLC. Furthermore, a solution of CA was prepared in
water at a concentration of 40 µg/mL and exposed to UV light. Aliquots of this solution were withdrawn at 2, 4 and 8 h and analyzed
immediately via HPLC.
Accuracy
The accuracy of the method was determined by measuring the reference standard recovery in triplicate at three levels from 80 to 120%
of the method concentration (40 µg/mL), according to ICH recommendations. A standard stock solution containing 100 µg/mL of CA
was prepared in ethanol and purified water (40:60 v/v). In 10 mL volumetric flasks; 1.2, 2.0 and 2.8 mL aliquots of this standard solution
(concentrations of 12, 20 and 28 µg/mL, respectively) were individually added to 2.0 mL sample solutions at 100 µg/mL (concentration of
20 µg/mL). The final concentrations were 32, 40 and 48 µg/mL, which
correspond to 80, 100 and 120% of the target concentrations, respectively. The mean recoveries, expressed in terms of the percentage
307
Validation of HPLC–UV Assay of Caffeic Acid in Emulsions
Table I. Methods for the Determination of CA by HPLC
Conditions
Retention
time (min)a
Detection
system
Matrices
Reference
Kingsorb column C18 (150 × 4.6 mm, 5 µm). 0.1%
orthophosphoric acid in water (v/v) (eluent A) and 0.1%
orthophosphoric acid in methanol (v/v) (eluent B)
Hypersil ODS column (250 × 4.0 mm, 5 µm). The mobile phase
was acetonitrile–water (18:82) containing 2% acetic acid. The
flow rate was set at 1.0 mL/min
Column C-8 reverse phase (250 mm × 3 mm, 5 µm). The mobile
phase was acetonitrile–water (18:80) with 2% acetic acid and a
flow rate of 0.5 mL/min
C18 column (50 mm × 2.1 mm, 3 µm particle size, Alltech,
Deerfield, IL, USA). Flow rate was set to 0.2 mL/min, and the
gradient used was as follows (where A = 1% acetic acid in
nano-pure water and B = HPLC grade acetonitrile): t = 0–4 min:
90% A (10% B); t = 4–15 min: 90–60% A (10–40% B); t = 15–
30 min: 60–40% A (40–60% B); t = 30.1–35 min: 0% A (100%
B); t = 35.1–43 min: 90% A (10% B)
Column Luna C18 (250 × 4.6 mm × 5 μm); mobile phase A: MeOH
0.1% formic acid, B: H2O + 0.1% formic acid). Gradient 5–45%
of A to B at 190 min
Intersil C8–3 column (5 mm; 250 × 2.1 mm) with acetonitrile/
0.05% triethylamine solution (70:30, v/v) as mobile phase at a
flow rate of 0.2 mL/min
IB-SIL RP 18 (250 × 4.6 mm × 5 μm) Phenomenex. Mobile phase:
solvent
A: water/AcOH 98:2; solvent B: MeOH/AcOH 98:2. Gradient:
15% B to 40% B, 30 min; 40% B to 75% B, 10 min; 75% B to
85% B, 5 min. Flow rate: 1.2 mL/min
Phenomenex (Torrance, CA, USA) C18
Synergi 4 µm Hydro-RP 80 Å pore size (150 × 3.0 mm) column
fitted with a Phenomenex (Torrance, CA, USA) security guard
column (4 × 3.0 mm). The mobile phase consisted of 2% (v/v)
acetic acid in water (eluent A) and of 0.5% acetic acid in
water and methanol (10/90, v/v; eluent B). The flow rate was
0.4 mL/min
A Chromolith RP-18 column (Inertsil 7 ODS-3, 4.6 mm internal
diameter, 250 mm; Merck). Mobile phase was 80% solution A
(diluted with water containing 1% acetic acid) and 20%
acetonitrile. The flow rate of the mobile phase was 1 mL/min
ProStar 500 column valve module was used with 0.1% aqueous
formic acid:methanol gradient mobile phase
Spherigel analytical column (250 × 4.6 mm) packed with 5 mm C18
silica. The gradient elution was programmed as follows: 0–9 min:
10–18.5% B; 9–9.5 min: 18.5–45% B; 9.50–39.50 min:
45–80% B; 39.5–42.0 min: 80–100% B; 42.0–45.0 min: 100–
10% B. The flow rate was 1 mL/min
3.5
UV at
330 nm
Rosemary leaves
(18)
NI
UV at
302 nm
Receptor solution in a permeation
study
(19)
6.0
UV at
330 nm
Receptor solution in a permeation
study
(20)
NI
ESI–MS
Echinacea purpurea extracts
(21)
3.7
UV/MS
Myrcia bella hydroalcoholic extract
(22)
4.0
MS
Rat plasma
(23)
12.4
UV at
325 nm
Leaves of I. paraguariensis and Ilex
spp.
(24)
30
DAD at
293 nm
Juices of Punica granatum L.
(25)
7.75
UV at
280 nm
Wheat germ oil, peanut oil, potato
plants, curly kale, JuZenTa and O.
gratissimum
(26)
14.3
UV at
325 nm
UV at
329 nm
Dipsacus asperoides (dried raw herb)
(27)
Roots and extracts of Echinacea
purpurea
(28)
11.12
NI, not informed; UV, ultraviolet; MS: mass spectroscopy; ODS, octadecylsilane.
Table II. Percentage Composition of the Proposed Emulsion
INCI name
%
Cetearyl alcohol
Ceteareth-20
Ethylhexyl stearate
Glyceryl stearate
Propylene glycol
Disodium EDTA
Methylparaben
Propylparaben
Sodium polyacrylate (Rapithix A-100)
Water
4.0
2.0
1.5
1.0
3.0
0.05
0.18
0.02
1.5
q.s.p. 100%
recovery of CA of the assay and the relative standard deviation (RSD),
were determined.
Precision
The precision was evaluated in terms of the repeatability and intermediate precision. The repeatability was evaluated by analyzing CA
working standard solutions at the same concentration and during
the same day. The intermediate precision was studied by repetition
of the assays on two different days by two analysts. Seven replicates
at a concentration of 40 µg/mL were prepared and assayed. The absorbance data at 325 nm were analyzed. The percentage RSD of the analytical responses was calculated.
308
Spagnol et al.
Robustness
The robustness of the method was evaluated by changing the established chromatographic conditions. Standard solutions of CA at
40 µg/mL were injected into the HPLC with variation of the following
variables:
•
Wavelength: 323, 325 and 327 nm.
Flow rate of mobile phase: 0.65, 0.70 and 0.75 mL/min.
Proportion of mobile phase: ethanol and water 35:65, 40:60 and
45:55.
Injection volume: 8, 10 and 12 μL.
Ethanol manufacturer: JT Baker® and Tedia®.
•
•
•
•
The results were evaluated by considering the RSD. The parameter
that presents the greatest variation in the measurements should be controlled and monitored during routine analysis.
Limits of detection and quantification
The limit of detection (LOD) and limit of quantification (LOQ) of the
method were obtained from Equations (1) and (2):
LOD ¼ 3 SD/α
ð1Þ
LOQ ¼ 10 SD/α;
ð2Þ
Results
Method validation
The analytical parameters, i.e., linearity, specificity, LOD, LOQ, precision, accuracy and robustness were evaluated to validate the method,
according to ICH recommendations (31).
Linearity
A working standard solution of CA (200 µg/mL) was appropriately diluted with the diluent solution to obtain solutions in the concentration
range of 10–60 µg/mL CA. Ten microliters of each solution were injected in triplicate into the column under the previously described operating chromatographic conditions. The equation of the line
determined by the least squares method is: y = 158527.8137x –
2234.0148, with a correlation coefficient (r) greater than 0.9999.
The data were validated by means of an ANOVA test (Table III),
which showed significant linear regression (Fcalculated > Fcritical, P =
5%) and no significant lack of fit (Fcalculated < Fcritical, P = 5%). Figure 3
shows the chromatograms obtained with six concentrations of CA
with a retention time of 5.70 min, and Figure 4 shows the overlap of
the chromatograms of the CA standard and the sample, and the emulsion without CA.
where SD is the standard deviation of the intersection and α is the average slope, obtained from calibration curves of the linearity study.
LOD and LOQ
The LOD and LOQ were determined to be 1.44 and 4.38 µg/mL, respectively. The low values indicate the sensitivity of the method.
Table III. Linearity Parameters for Determination of CA and
Summary of ANOVAa
Specificity
The specificity of the method was checked by comparing the chromatograms obtained for pure CA solution in the forced degradation
procedure. The specificity of the method can be determined by addition of impurities and degradation products, obtained experimentally
or by inducing their formation (32). The CA solutions were subjected
to degradation in acidic, alkaline, oxidative, aqueous and photolytic
media.
After the exposure period, the solutions were immediately injected
into the HPLC and the obtained chromatograms are presented in
Figure 5. No interfering peaks were observed in the CA analysis,
i.e., no peaks were observed near the standard retention time of CA.
Parameter
325 nm
Linearity range (µg/mL)
Slope
Intercept
Correlation coefficient (r)
Regression
Lack of fit
10–60
158,527
2,234
0.9999
8677 (4.75)
0.02 (3.26)
a
Values are reported as the mean of three calibration curves generated on
three consecutive days (n = 3).
Figure 3. Chromatogram of CA at six concentrations.
309
Validation of HPLC–UV Assay of Caffeic Acid in Emulsions
Figure 4. Overlapped chromatograms of CA standard and the sample and chromatogram of emulsion without CA.
Figure 5. Chromatograms of the degradation products of CA generated by acid (A), photolytic (B), neutral (C), oxidative (D) and basic (E, F) treatment.
Precision
The precision was determined based on the repeatability and the RSD
was calculated as 1.95%. The repeatability demonstrates the correlation between results acquired within a short period of time by the same
analyst using the same instrumentation.
We also evaluated the precision by comparison of the results obtained by two different operators, which yielded a RSD of 1.45%
for the second analyst. The F-test showed no significant difference
between the absorbance values determined by the two analysts since
Fcal 1.79 < Ftab 4.28 and Pvalue 0.25 > 0.05, proving the intermediate
precision of the proposed method, as shown in Tables IV and V.
Accuracy
The accuracy of the method was confirmed by determining the average
recoveries of the samples by applying the standard addition method.
As shown in Table VI, the mean percentage recoveries of the 40 µg
CA product were found in accordance with the fixed limits of
310
Spagnol et al.
Table IV. Precision of Method for CA Analysis
40 µg/mL
Area 1
Area 2
Area 3
Area 4
Area 5
Area 6
Area 7
Average
RSD%
Analyst 1
Analyst 2
5,871,711
5,751,237
5,762,469
5,790,979
5,505,668
5,771,495
5,672,982
5,684,689
5,708,533
5,744,990
5,655,685
5,545,226
5,717,722
5,734,227
5,699,252
5,717,549
1.95
1.45
Table V. F-Test of Intermediate Precision of the HPLC Method
Average
Variance
Observations
Gl
F
P(F ≤ f ) one-tailed
F crit
Analyst 1
Analyst 2
5,699,252.85
8.9619E–05
7
6
1.79978217
0.246407065
4.283865714
5,717,549.00
3.79048E–05
7
6
Table VII. Values Obtained in the Evaluation of the Robustness of the
HPLC Protocol for AC Analysis
Variable
Range
investigated
CA (%)
RSD (%)
Wavelength (nm)
323
325
327
0.65
0.70
0.75
35:65
40:60
45:55
8
10
12
JT Baker®
Tedia®
98.57
100.00
99.02
103.45
100.00
100.82
94.36
100.00
96.40
99.32
100.00
98.11
100.00
96.02
0.7375
Mobile phase flow (mL/min)
Ethanol/water ratio in the mobile
phase
Table VI. Accuracy of Method for Analysis of CA emulsion
R1
R2
R3
CA added
(µg/mL)
CA found
(µg/mL)
Recovery
(%)
Mean
recovery
(%)
RSD (%),
n=3
32
40
48
32.16
39.92
48.27
100.51
99.80
100.57
100.29
0.43
Injection volume (μL)
Manufacturer of ethanol
98.0–102.0%, indicating the suitability of the developed method for
quantifying the concentration of CA in emulsions.
Robustness
The robustness of the method was evaluated by modifying the mobile
phase flow, wavelength, ethanol/water ratio in the mobile phase, injection volume and manufacturer of the solvent used. The RSD of the data
obtained after these changes did not exceed 5%. This indicates that the
method is robust, since small changes in the method used did not cause
significant variations in the results, as presented in Table VII.
Discussion
The method was developed and validated using a mixture of water and
ethanol as the solvent, and this is the first demonstration of the use of
this mixture for the determination of CA, as can be seen in Table I. The
difficulty in this method is to find a balance between the mobile phase
flow, ethanol/water ratio in the mobile phase and the pressure of the
equipment. Successfully overcoming this difficulty and finding a balance in these parameters could facilitate harnessing of the advantages
of this technique, since this method uses only green solvents and reduces the generation of toxic waste. The developed method is based on the
high absorption intensity of the functional chromophore groups present in CA in this solvent system and presents advantages such as simple sample preparation, low cost and easy waste disposal.
The results obtained in the evaluation of the linearity demonstrate
linear correlation between the absorbance of the CA solutions and the
concentrations in the range evaluated. Plots of the absorbance versus
concentration were linear in the concentration range of 10–60 µg/mL.
The equation of the line for the method is y = 158527.8137× –
2234.0148, with a correlation coefficient (r) greater than 0.9999.
1.7776
2.9481
0.9638
2.8655
RSD, relative standard deviation.
The limits of detection and quantification were calculated based on
the calibration curves generated on different days. The detection and
quantitation limits were 1.44 and 4.38 µg/mL, respectively, which
demonstrates that the range used is above the limits and, thus, the concentration values were appropriate. The precision of the method was
demonstrated by analysis of the repeatability (intra-day precision) and
intermediate precision (inter-day and between-analysts). The RSD of
the experimental value obtained from intra-day analysis was 1.95%
for analysis of seven replicates of CA performed on the same day
under the same conditions. In the analysis of the inter-day precision,
the analyses were performed on different days, yielding an average
content of 100.32% and an RSD of 1.95%.
The accuracy was evaluated by the addition method and calculated
mathematically based on the sample recovery. The results obtained are
in the range of 99.8–100.57%, which is consistent with the recommended range for the accuracy of analytical methods (98–102%)
(33). The parameters chosen to evaluate the robustness of the method
were the mobile phase flow, wavelength, ethanol/water ratio in the
mobile phase, injection volume and manufacturer of the solvent
used. The RSD values of the analytical results obtained after these
changes did not exceed 5%. This indicates that the method is robust,
since small changes in the method used did not cause significant variations of the results. The proposed HPLC–UV method meets all the
requirements of the official codes agency guidelines (31, 33, 34), making it adequate, presenting simplicity, specificity, linearity, precision,
accuracy and robustness, and useful for assay of CA in emulsions.
Conclusion
An analytical HPLC–UV method was developed for the quantitative
determination of CA in emulsions. The advantages of this method
over other existing methods include its simplicity, speed and low
Validation of HPLC–UV Assay of Caffeic Acid in Emulsions
cost. The results indicate that the developed HPLC–UV method has
good linearity, selectivity, accuracy, precision and robustness and adequate detection and quantification limits. Therefore, the validated
method can be easily applied to routine analysis of CA.
Acknowledgments
We thank FAPESP, CNPq and PADC-FCF-UNESP for their financial support.
References
1. Magnani, C., Isaac, V.L.B., Corrêa, M.A., Salgado, H.R.N.; Caffeic acid: a
review of its potential use in medications and cosmetics; Anal. Methods,
(2014); 6: 3203–3210.
2. Clifford, M.N.; Chlorogenic acids and other cinnamates: nature, occurrence, dietary burden, absorption and metabolism; J. Sci. Food Agric.,
(2000); 80: 1033–1043.
3. Lustosa, S.R., Galindo, A.B., Nunes, L.C.C., Randau, K.P., Rolim Neto, P.
J.; Propolis: Atualizações sobre a química e a farmacologia; Rev. Bras.
Farmacogn., (2008); 18: 447–454.
4. Huang, M.T., Ferraro, T.; Phenolic-compounds in food and cancer prevention; ACS Symp. Ser., (1992); 507: 8–34.
5. Greenwald, P.; Clinical trials in cancer prevention: current results and perspectives for the future; J. Nutr., (2004); 134: 3507S–3512S.
6. Sanchez-Moreno, C., Jimenez-Escrig, A., Saura-Calixto, F.; Study of lowdensity lipoprotein oxidizability indexes to measure the antioxidant activity
of dietary polyphenol; Nutr. Res., (2000); 20: 941–953.
7. Vinson, J.A., Teufel, K., Wu, N.; Red wine, dealcoholized red wine, and
especially grape juice, inhibit atherosclerosis in a hamster model;
Atherosclerosis, (2001); 156: 67–72.
8. Worldwide Chemical Information, Trading & Advertising, http://www.
chemicalland21.com/lifescience/phar/caffeic%20acid.htm (accessed February 1, 2014).
9. Gennaro, A.R.; Remington the science and practice of pharmacy, 20th ed.
Lippincott Williams & Wilkins, Philadelphia, (2000).
10. Smith, E.W., Malbach, H.I., Surber, C.; Use of emulsions as topical drug
delivery systems. In Niedelloud, F., Marti-Mestres, G. (eds). Pharmaceutical
emulsions and suspensions. Marcel Dekker, New York, (2000), pp. 259–269.
11. Fonseca, Y.M.; Desenvolvimento de formulações tópicas contendo extrato
de própolis verde: Estudos de estabilidade, liberação, permeação e retenção
cutânea. Tese de doutorado. Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, Ribeirão Preto, (2007).
12. Silva, E.C., Soares, I.C.; Tecnologia de emulsões; Cosmet Toiletries, (1996);
8: 37–46.
13. Prista, L.N., Alves, A.C., Morgado, R.; Tecnologia farmacêutica, 5th ed.
Fundação Calouste Gulbenkian, Lisboa, (2008).
14. Corrêa, M.A.; Cosmetologia: Ciência e Técnica, Medfarma, São Paulo,
(2012).
15. Mostefa, N.M., Sadok, A.H., Sabri, N., Hadji, A.; Determination of optimal cream formulation from long-term stability investigation using a surface response modeling; Int. J. Cosmet. Sci., (2006); 28: 211–218.
16. Isaac, V.L.B., Cefali, L.C., Chiari, B.G., Oliveira, C.C.L.G., Salgado, H.R.N.,
Corrêa, M.A.; Protocolo para ensaios físico-químicos de estabilidade de fitocosméticos; Rev. Cienc. Farm. Básica Apl., (2008); 29: 85–100.
311
17. La Roca, M.F., Sobrinho, J.L.S., Nunes, L.C.C., Neto, P.J.R.; Desenvolvimento e validação de método analítico: Passo importante na produção de
medicamentos; Rev. Bras. Farm., (2007); 88: 177–180.
18. Wang, H., Provan, G.J., Helliwell, K.; Determination of rosmarinic acid and
caffeic acid in aromatic herbs by HPLC; Food Chem., (2004); 87: 307–311.
19. Saija, A., Tomaino, A., Trombetta, D., De Pasquale, A., Uccella, N.,
Barbuzzi, T., et al.; In vitro and in vivo evaluation of caffeic and ferulic
acids as topical photoprotective agents; Int. J. Pharm., (2000); 199: 39–47.
20. Marti-Mestres, G., Mestres, J.P., Bres, J., Martin, S., Ramos, J., Viana, L.;
The in vitro percutaneous penetration of three antioxidant compounds;
Int. J. Pharm., (2007); 331: 139–144.
21. Cech, N.B., Eleazer, M.S., Shoffner, L.T., Crosswhite, M.R., Davis, A.C.,
Mortenson, A.M.; High performance liquid chromatography/electrospray
ionization mass spectrometry for simultaneous analysis of alkamides and
caffeic acid derivatives from Echinacea purpurea extracts; J. Chromatogr.
A, (2006); 1103: 219–228.
22. Saldanha, L.L., Vilegas, W., Dokkedal, A.L.; Characterization of flavonoids
and phenolic acids in Myrcia bella Cambess using FIA-ESI-IT-MSn and
HPLC PAD-ESI-IT-MS combined with NMR; Molecules, (2013); 18:
8402–8416.
23. Guy, P.A., Renouf, M., Barron, D., Cavin, C., Dionisi, F., Kochhar, S., et al.;
Quantitative analysis of plasma caffeic and ferulic acid equivalents by liquid
chromatography tandem mass spectrometry; J. Chromatogr. B: Anal.
Technol. Biomed. Life Sci., (2009); 877: 3965–3974.
24. Filip, R., L’opez, P., Giberti, G., Coussio, J., Ferraro, G.; Phenolic compounds in seven South American Ilex species; Fitoterapia, (2001); 72:
774–778.
25. Fischer, U.A., Carle, R., Kammerer, D.R.; Identification and quantification
of phenolic compounds from pomegranate (Punica granatum L.) peel, mesocarp, aril and differently produced juices by HPLC–DAD–ESI/MSn; Food
Chem., (2011); 127: 807–821.
26. Ye, J.-C., Hsiao, M.-W., Hsieh, C.-H., Wu, W.C., Hung, Y.-C., Chang, W.C.;
Analysis of caffeic acid extraction from Ocimum gratissimum Linn. by high
performance liquid chromatography and its effects on a cervical cancer cell
line; Taiwan J. Obstet. Gynecol., (2010); 49: 266–271.
27. Pearson, J.L., Lee, S., Suresh, H., Low, M., Nang, M., Singh, S., et al.; The
liquid chromatographic determination of chlorogenic and caffeic acids in
Xu Duan (Dipsacus asperoides) raw herb; Anal. Chem., (2014); 2014: 1–7.
28. Luo, X.B., Chen, B., Yao, S.-Z., Zeng, J.-G.; Simultaneous analysis of caffeic acid derivatives and alkamides in roots and extracts of Echinacea purpurea by high-performance liquid chromatography–photodiode array
detection–electrospray mass spectrometry; J. Chromatogr. A, (2003); 986:
73–81.
29. Brazilian Pharmacopeia, 5th ed. Anvisa, Brasília, Brazil, (2010).
30. Kogawa, A.C., Salgado, H.R.N.; Quantification of doxycycline hyclate in
tablets by HPLC-UV method; J. Chromatogr. Sci., (2012); 5: 1–7.
31. ICH. (2005) International Conference on Harmonization: Validation of analytical procedures: Text and Methodology Q2(R1). http://www.ich.org/
LOB/media/MEDIA417.pdf (accessed September 7, 2014).
32. Ermer, J.; Validation in pharmaceutical analysis. Part I: an integrated approach; J. Pharm. Biomed. Anal., (2001); 24: 755–767.
33. Brazil, Ministério da Saúde. Resolução RE no 899, de 29 de maio de 2003.
Guia para validação de métodos analíticos e bioanalíticos. Diário Oficial da
União, Brasília, (2003).
34. USP. The United States Pharmacopeia, 33rd ed. The United States Pharmacopeial Convention, Rockville, (2010).